University  of  Cal 

..em  California  Labor;.' 

^p€- -  LiuivJjTV-  I  -sued  August  18,  1909. 

0^        U.  S.  DEPARTMENT  OF  AGRICULTURE, 

Q  ^^  BUREAU  OF  ANIMAL  INDUSTRY.— BULLETIN  116. 

8   -  A.  D.  MKLVIN,  CHIEF  OP  BUREAU. 


.ESTS  CONCERNING  TUBERCLE  BACILLI 

IN  THE  CIRCULATING  BLOOD. 

University  of  California 


BY 


Southern  California  Laboratory 

OF  — 

Plant  Pathology 


E.  C.  SCHROEDER,  M.  D.  V., 

C^  Superintendent  of  Experiment  Station, 

3  ^  AND 

OT    3' 

W.  E.  COTTON, 

CT*  r> 

Expert  Assistant  at  Experitnent  Station. 
P     0>   .- 


o  tr. 

3      H-b 

»    O 
"   g^ 

5' 


WASHINGTON: 

GOVERNMENT   PRINTING   OFFICE. 

1909. 


Issued  August  18,  1909. 

U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— BULLETIN  116. 

A.  D.  MRLVIN,  CHIEF  OF  BUREAU. 


TESTS  CONCERNING  TUBERCLE  BACILLI 
IN  THE  CIRCULATING  BLOOD. 


BY 


E.  C.  SCHROEDER,  M.  D.  V., 

Superintendent  of  Experiment  Station, 
AND 

W.    E.    COTTON. 
Expert  Assistant  at  Experiment  Station. 


WASHINGTON: 

GOVERNMENT    PRINTING   OFFICE. 

1909. 


THE  BUREAU  OF  ANIMAL  INDUSTRY. 


Chief:  A.  D.  MELVIN. 
Assistant  Chief:  A.  M.  FARRINGTON. 
Chief  Clerk:  CHARLES  C.  CARROLL. 
Biochemic  Division:  M.  DORSET,  chief. 
Dairy  Division:  B.  H.  RAWL,  chief. 

Inspection  Division:  RICE  I*.  STEDDOM,  chief;  MORRIS  WOODEN,  R.  A.  RAMSAY, 
and  ALBERT  E.  BEHNKE,  associate  chiefs. 
Pathological  Division:  JOHN  R.  MOHLER,  chief. 
Quarantine  Division:  RICHARD  W.  HICKMAN,  chief. 
Zoological  Division:  B.  H.  RANSOM,  chief. 
Experiment  Station:  E.  C.  SCHROEDER,  superintendent. 
Animal  Husbandman:  GEORGE  M.  ROMMEL. 
Editor:  JAMES  M.  PICKENS. 
2 


LETTER  OF  TRANSM1TTAL. 


U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY, 

Washington,  D.  C.,  July  7,  '1909. 

SIR:  I  have  the  honor  ^o  transmit  herewith,  and  to  recommend 
for  publication  in  the  bulletin  series  of  this  Bureau,  the  accompany- 
ing manuscript,  entitled  "Tests  Concerning  Tubercle  Bacilli  in  the 
Circulating  Blood,"  by  Dr.  E.  C.  Schroeder  and  Mr.  W.  E.  Cotton, 
of  the  Experiment  Station  of  this  Bureau. 

The  tests  described  in  the  bulletin  were  undertaken  upon  the 
appearance  of  a  recent  paper  in  which  were  recorded  a  large  number 
of  microscopic  examinations  of  the  blood  of  tuberculous  individuals, 
the  result  of  which,  it  was  stated,  proved  that  tuberculosis  in  all  its 
forms  was  a  bacteriemia. 

This  conclusion  was  entirely  contrary  to  the  views  of  the  authors 
of  this  bulletin,  based  upon  their  work  in  the  Bureau  for  a  long  series 
of  years;  and  as  the  matter  was  of  great  importance  in  its  bearing  on 
the  tuberculosis  question,  it  was  considered  advisable  to  put  it  to  a 
thorough  test.  Accordingly  inoculation  experiments  were  made  with 
the  blood  of  a  large  number  of  tuberculous  cattle,  and  the  results 
have  demonstrated  that  tuberculosis  is  not  in  any  sense  a  bacteriemia, 
and  that  if  tubercle  bacilli  ever  float  in  the  blood  of  tuberculous 
animals  this  is  an  exceedingly  rare  condition. 
Respectfully, 

A.  D.  MELVIX, 

Chief  of  Bureau. 
Hon.  JAMES  WILSON. 

Secretary  of  Agriculture. 

3 


CONTENTS. 


Paga 

Introduction 7 

Summary  of  present  tests 9 

Records  of  rattle  and  results  of  guinea-pig  inoculations 10 

Discussion  of  results 18 

Supplemental  tests  regarding  possible  immunity 20 

Conclusions..                                                                     • 23 


TESTS  CONCERNING  TUBERCLE  BACILLI  IN  THE  CIR- 
CULATING BLOOD. 


INTRODUCTION. 

In  a  paper  dealing  with  the  occurrence  of  tubercle  bacilli  in  the 
circulating  blood,  read  before  a  medical  society  some  months  since 
and  soon  afterwards  published  in  a  medical  journal."  there  were  re- 
corded the  results  of  microscopic  examinations  of  the  blood  of  125 
tuberculous  individuals,  some  of  whom  were  affected  with  only 
incipient  tuberculosis,  and  the  statement  was  made  that  tubercle 
bacilli  were  found  in  the  blood  of  even-  one  of  them.  In  some  cases 
only  a  few  bacilli  were  seen,  but  u  thev  were  mostly  in  large  mim- 

v  */  »/  O 

bers,  and  clumps  of  30  to  40  bacilli  were  not  unusual,  especially  in 
cases  of  acute  miliary  tuberculosis."  From  his  observations  the 
author  of  that  paper  formulated  the  conclusion :  "  It  appears  that 
tuberculosis  in  all  its  forms  is  a  bacteriemia." 

That  tubercle  bacilli  occasionally  float  in  the  blood  stream  is  hardly 
open  to  question,  because  of  the  occurrence  of  isolated  lesions  in  the 
bodies  of  otherwise  tuberculous  as  well  as  otherwise  healthy  indi- 
viduals located  in  regions  remote  from  the  various  channels  that  com- 
municate with  the  exterior.  The  same  is  true  when  we  consider  cases 
of  more  or  less  generalized  tuberculosis  with  many  lesions  in  widely 
separated  portions  of  the  body  and  cases  of  miliary  tuberculosis  with 
innumerable  lesions  of  approximately,  if  not  precisely,  the  same  age 
and  stage  of  development.  But  such  occasional  presence  of  tubercle 
bacilli  in  the  circulating  blood  is  a  very  different  condition  from  their 
constant  occurrence  in  it  in  sufficient  numbers  to  justify  the  classifi- 
cation of  tuberculosis  as  a  bacteriemia.  Hence.  Rosenberger's  con- 
clusion was  received  with  considerable  surprise  and  doubt. 

Although  the  conclusion  seemed  sufficiently  incredible  because  of 
the  simple  fact  that  a  constant  occurrence  of  tubercle  bacilli  in  the 
blood  of  all  tuberculous  individuals  could  hardly  have  been  over- 
looked by  the  host  of  investigators  who  have  studied  tuberculosis 

a  Rosenberger.  Handle  C..  "The  presence  of  tubercle  bacilli  in  the  circulating 
blood  in  tuberculosis."  (Read  before  the  Pathological  Society  of  Philadelphia. 
December  10.  190X.)  American  Journal  of  the  Medical  Sciences,  vol.  137,  No. 
2.  pp.  267-269.  Philadelphia  and  New  York.  February.  1909. 


8  TESTS   CONCERNING   TUBERCLE   BACILLI    IN    THE   BLOOD. 

with  no  greatly  different  technique  than  Rosenberger  used,  we  did  not 
feel  warranted  in  opposing  it  without  offering  some  specific  evidence. 
Because  of  the  important  bearing  of  the  matter  on  the  tuberculosis 
problem,  the  experiments  hereinafter  reported  were  carried  out. 

Rosenberger  stated  that  he  found  tubercle  bacilli  on  microscopic 
examination  in  the  blood  of  every  one  of  the  125  cases  of  tuberculosis 
he  studied,  notwithstanding  that  some  of  the  cases  were  incipient  and 
failed  to  show  tubercle  bacilli  in  the  sputum.  It  was,  therefore, 
almost  taken  for  granted  that  the  microscopic  examination  of  blood, 
according  to  his  method,  of  animals  affected  wdth  advanced  and  long 
standing  tuberculosis  and  animals  that  were  expelling  tubercle  bacilli 
from  their  bodies  in  large  numbers  would  reveal  at  least  a  few 
tubercle  bacilli.  A  considerable  number  of  such  microscopic  examina- 
tion's were  made,  but  not  a  tubercle  bacillus  was  found  in  our  blood 
preparations,  and  hence  we  have  to  record  wholly  negative  results 
with  the  blood  of  tuberculous  animals.  Similar  negative  results  were 
obtained  with  the  blood  of  tuberculous  persons  in  two  large  New  York 
hospitals.0 

It  is  not  uncommon  for  virulent  tubercle  bacilli  to  be  present  in 
animal  substances  in  numbers  too  small  to  serve  for  their  detection  by 
optical  methods.  For  example,  at  the  Experiment  Station  we  found 
the  intraabdominal  injection  of  guinea  pigs  with  suspected  milk  to 
be  a  test  for  tubercle  bacilli  that  has  fully  fifty  times  the  delicacy  of 
a  microscopic  examination.  Furthermore,  tinctoral  and  optical  meth- 
ods of  distinguishing  between  tubercle  bacilli  and  other  acid-fast 
bacteria  are  not  wholly  satisfactory,  hence  we  concluded  to  inject  a 
sufficient  number  of  guinea  pigs  \vith  blood  from  a  sufficient  number 
of  certainly  tuberculous  cattle  to  show  conclusively  that  tubercle 
bacilli  either  are  or  are  not  commonly  present  in  such  blood. 

Incidently  it  appears  that  Doctor  Rosenberger  failed  to  confirm 
adequately  by  animal  experiments  his  surprising  microscopic  observa- 
tions, which,  if  correct,  would  have  been  of  the  greatest  value  alone  for 
the  early  and  certain  diagnosis  of  tuberculosis.  In  all  he  inoculated 
only  two  guinea  pigs,  of  which  he  gives  a  record;  one  with  blood 
from  a  tuberculous  person  who  was  expelling  tubercle  bacilli  per 
rectum,  and  one  with  blood  from  a  case  of  acute  miliary  tuberculosis. 
The  development  of  tuberculosis  in  the  latter  guinea  pig  can  not 
be  regarded  as  a  remarkable  phenomenon.  There  is  nothing  about 
the  fact  that  a  guinea  pig  contracted  tuberculosis  after  an  injection 
of  blood  obtained  from  a  case  of  acute  miliary  tuberculosis  that 
necessitates  a  modification  of  our  currently  accepted  views  on  the 
presence  of  tubercle  bacilli  in  the  circulating  blood.  That  is  to  say, 
we  need  not  look  upon  tuberculosis  as  a  bacteriemia  because  tubercle 

0  Editorial  in  the  Medical  Record,  New  York.  April  3,  1909.  p.  568. 


CONDITION  OF  CATTLE  USED  IN  TESTS.  9 

bacilli  were  demonstrated  in  blood  of  a  kind  in  which  we  have  long 

O 

considered  that  they  might  sometimes  occur. 

This  leaves  one  guinea  pig  that  may  have  some  evidential  value, 
but  we  must  not  lose  sight  of  the  fact  that  it  was  injected  with 
blood  obtained  from  a  person  who  was  expelling  tubercle  bacilli  from 
his  body  and  hence  to  some  extent  infecting  his  environment.  We 
must  also  bear  in  mind  that  guinea  pigs  are  highly  susceptible  to 
tubercle  bacilli  injected  into  their  bodies,  and  that  it  is  often  impos- 
sible for  an  investigator  who  handles  much  tuberculous  material, 
who  is  in  frequent  contact  with  tuberculous  persons,  and  whose  envi- 
ronment may  be  characterized  as  containing  tubercle  bacilli,  to  elimi- 
nate all  danger  of  extraneous  tuberculous  infection  sufficiently  to 
make  a  test  satisfactory  when  he  seeks  to  verify  the  tuberculous 
character  of  some  material  from  a  tuberculous  individual  by  the 
injection  of  one,  and  only  one,  guinea  pig. 

SUMMARY  OF  PRESENT  TESTS. 

Our  own  tests  were  made  entirely  with  the  blood  of  tuberculous 
cattle.  In  every  case  the  blood  was  drawn  from  the  jugular  vein  of 
the  tuberculous  animal  and  injected  in  its  fresh,  naturally  warm 
state  into  the  peritoneal  cavity  of  a  guinea  pig.  The  tuberculous 
cattle,  as  their  records  show,  may  be  divided  into  four  distinct  lots, 
according  to  their  tuberculous  condition : 

Lot  1.  Four  cattle,  the  precise  tuberculous  condition  of  which  is 
known,  because  they  were  killed  and  examined  post-mortem  shortly 
after  blood  was  drawn  from  them  for  guinea-pig  injections. 

Lot  2.  Six  cattle,  known  to  be  tuberculous  because  they  had  re- 
acted with  tuberculin,  because  tubercle  bacilli  were  found  in  their 
feces  on  microscopic  examination,  and  because  their  feces  were  proven 
to  be  infectious  by  animal  experiments. 

Lot  3.  Nineteen  cattle,  known  to  be  tuberculous  because  they  had 
reacted  with  tuberculin  and  because  tubercle  bacilli  were  found  in 
their  feces  on  microscopic  examination. 

Lot  4.  Thirteen  cattle,  known  to  be  tuberculous  because  they  had 
reacted  with  tuberculin. 

We  made  no  attempt  to  treat  the  blood  used  for  the  injections  in 
any  way,  because  we  assumed  that  the  best  results  would  be  obtained 
with  it  by  transferring  it  as  rapidly  as  possible  from  the  tuberculous 
cattle  to  the  peritoneal  cavities  of  the  guinea  pigs.  It  was  learned 
from  the  injections  that  guinea  pigs  tolerate  a  relatively  large  quan- 
tity of  bovine  blood  in  their  peritoneal  cavity.  The  guinea  pigs  that 
died  shortly  after  as  the  result  of  the  blood  injections  (about  15  per 
cent  of  all  injected)  with  few  exceptions  showed  extreme  impaction 
and  some  inflammation  of  the  large  bowel,  associated  in  several 
instances  with  imagination  of  the  colon. 
2279— Bull.  116—09 2 


10  TESTS   CONCEENING   TUBERCLE   BACILLI   IN    THE  BLOOD. 

The  possibility  exists  that  the  intraperitoneal  injection  of  from 
3  to  5  cubic  centimeters  of  fresh,  warm1  blood  from  tuberculous  cattle 
induces  an  immunity  in  guinea  pigs  to  the  tubercle  bacilli  the  blood 
may  contain.  Though  this  view  is  purely  hypothetical  and  we  know 
of  nothing  to  sustain  it,  we  have  carried  out  an  investigation  to  prove 
or  disprove  it,  and  will  give  the  results  later  in  this  paper. 

The  total  number  of  cattle  from  which  blood  injections  were  made 
was  42,  and  these,  as  their  records  show,  represent  a  considerable 
variety  relative  to  the  severity  and  extent  of  the  tuberculous  disease 
with  which  they  were  affected.  They  ranged  from  animals  that 
would  not  have  been  suspected  to  be  diseased  without  a  tuberculin 
test  to  a  cow  so  badly  affected  that  a  calf  of  which  she  became  the 
mother  a  little  less  than  a  year  before  her  blood  was  used  for  guinea- 
pig  injections  was  born  affected  with  tuberculosis  contracted  from 
ante-partum  exposure  to  her  tuberculous  body. 

The  total  number  of  guinea  pigs  injected  was  104.  Of  these,  16 
died  writhin  a  few  days  after  the  injection  and  no  doubt  as  a  result  of 
it.  Three  died  of  intercurrent  affections,  but  not  until  a  sufficient 
period  of  time  had  passed  for  lesions  of  tuberculosis  to  become 
clearly  manifest.  The  remaining  85  lived  until  they  were  killed 
after  a  lapse  of  from  seven  and  one-half  to  eleven  weeks,  or  an 
average  for  all  of  seventy  days  after  they  were  injected.  The  three 
guinea  pigs  that  died  of  intercurrent  affections  showed  no  lesions  of 
tuberculosis  on  post-mortem  examination,  and  84  of  the  85  guinea 
pigs  that  lived  until  they  were  killed  showed  no  lesions  of  any  kind 
on  autopsy.  One  guinea  pig  of  the  85  showed  lesions  very  slightly 
resembling  tuberculosis,  but  these  were  proved  by  microscopic  exami- 
nations and  guinea-pig  inoculation  tests  to  be  free  from  tubercle 
bacilli. 

A  detailed  record  of  the  cattle  and  guinea  pigs  used  in  our  tests 
follows : 

RECORDS  OP  CATTLE  AND  RESULTS  OF  GUINEA-PIG  INOCULATIONS. 

The  42  cattle  included  in  the  records  below  are  all  that  were  avail- 
able for  this  investigation  among  the  tuberculous  cattle  kept  for 
various  purposes  at  the  Bureau  of  Animal  Industry  Experiment  Sta- 
tion. The  general  condition  of  the  cattle  is  briefly  defined  as  good, 
fairly  good,  fair,  or  poor;  and  as  these  terms  are  used  somewhat 
arbitrarily,  it  is  desirable  to  specify  more  precisely  what  they  are 
intended  to  convey.  The  word  "  good  "  is  used  in  connection  with 
cattle  that  were  really  to  all  appearances  in  excellent  physical  condi- 
tion;  and  of  which  no  one  would  suspect  that  they  were  diseased. 
The  words  "  fairly  good  "  are  used  to  mean  that  condition  commonly 
found  among  dairy  cows  of  the  better  class.  "  Fair  "  is  used  to  desig- 


RESULTS   OF   GUINEA-PIG   INOCULATIONS.  11 

nate  a  condition  which  the  average  dairyman  regards  as  satisfactory, 
and  "  poor  "  is  applied  to  cattle  that  are  thin  or  that  show  visible 
symptoms  of  disease. 

LOT    1. 

Bull  393,  general  condition  very  good,  had  been  affected  with  tuberculosis  a 
year  or  more;  was  killed  and  examined  post-mortem  April  8,  1909.  The  autopsy 
revealed  only  one  small  tuberculous  lesion  located  in  one  of  the  superficial 
inguinal  glands. 

On  February  5,  1909,  two  guinea  pigs,  Nos.  2891  and  2892,  received  each  an 
intraabdominal  injection  of  2  c.  c.  of  blood  from  the  bull.  One  guinea  pig, 
No.  2891,  died  on  February  14,  1909,  affected  with  invagination  of  the  bowel. 
The  other  guinea  pig  remained  healthy  until  April  13,  1909  (sixty-seven  days 
after  injection),  when  it  was  killed  and  found  on  post-mortem  examination  to 
be  free  from  lesions  of  disease. 

Cow  533,  general  condition  poor,  had  been  affected  with  tuberculosis  two 
years  or  more;  was  killed  and  examined  post-mortem  April  24.  1909.  The 
autopsy  revealed  the  following  conditions:  The  principal  lobe  of  the  right  lung 
contained  a  cavity  about  3  inches  in  diameter,  partly  filled  with  pasty,  uecrotic 
tuberculous  material.  This  cavity  was  in  direct  communication  with  a  large 
bronchial  tube,  which  contained  a  considerable  amount  of  material  discharged 
from  the  cavity.  Sprinkled  throughout  the  lungs  generally  were  a  number  of 
smaller  tuberculous  foci,  in  a  completely  broken-down  condition.  The  medias- 
tiual  and  niesenteric  lymph  glands  and  the  liver  were  sprinkled  with  tuberculous 
foci,  some  of  which  were  as  much  as  half  an  inch  in  diameter.  Prior  to  the 
cow's  death  her  feces  were  examined  microscopically  on  nine  different  days  and 
on  six  of  these  days  were  found  to  contain  tubercle  bacilli. 

On  February  3,  1909,  two  guinea  pigs,  Nos.  2859  and  2SGO,  received  each  an 
intraabdominal  injection  of  3  c.  c  of  blood  of  the  cow.  The  guinea  pigs  re- 
mained healthy  until  April  13,  1909  (sixty-nine  days  after  injection),  when 
they  were  killed  and  found  on  post-mortem  examination  to  be  free  from  lesions 
of  disease. 

Cow  549,  general  condition  poor,  had  been  affected  with  tuberculosis  several 
years.  On  March  27,  1908,  she  gave  birth  to  a  calf  affected  with  congenital 
tuberculosis.  The  cow  was  killed  April  8.  1909,  and  on  autopsy  was  found  to 
be  affected  with  advanced,  generalized  tuberculosis.  The  lungs  contained 
lesions  varying  from  quite  recent  tuberculous  disease  to  large  tuberculous 
cavities  that  had  discharged  most  of  their  contents  through  the  bronchial  tubes. 
No  tests  were  made  relative  to  the  infectious  character  of  the  feces  before  death. 

On  February  3,  1909,  two  guinea  pigs,  Nos.  2863  and  2864,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  blood  from  the  cow.  The  guinea  pigs  were 
killed  April  13,  1909  (sixty-nine  days  after  injection),  and  on  post-mortem 
examination  were  found  to  be  free  from  lesions  of  disease. 

Cow  552,  general  condition  poor,  had  been  affected  with  tuberculosis  several 
years:  was  killed  April  1,  1909.  The  autopsy  revealed  a  fairly  generalized 
tuberculosis  with  lesions  of  greater  or  less  magnitude  in  the  lungs  and  in  the 
pharyngeal,  bronchial,  and  niesenteric  lymph  glands.  Prior  to  the  cow's  death 
her  feces  were  examined  microscopically  on  ten  different  days  and  on  six  of 
these  days  were  found  to  contain  tubercle  bacilli. 

Hogs  that  were  fed  feces  from  the  cow  contracted  tuberculosis,  and  guinea  pigs 
inoculated  subcutaueously  with  small  masses  of  her  feces  likewise  contracted 
tuberculosis. 


12  TESTS   CONCEKNING   TUBEECLE  BACILLI   IN   THE   BLOOD. 

Guinea  pigs  were  injected  intraabdominally  with  blood  from  this  cow  as 
follows : 

January  25,  1909,  guinea  pig  2785  received  5  c.  c. 

January  25,  1909,  guinea  pig  2786  received  5  c.  c. 

January  25,  1909,  guinea  pig  2783  received  2J  c.  c. 

January  25,  1909,  guinea  pig  2784  received  2J  c.  c. 

January  25,  1909,  guinea  pig  2781  received  1  c.  c. 

January  25,  1909,  guinea  pig  2782  received  1  c.  c. 

February  3,  1909,  guinea  pig  2861  received  3  c.  c. 

February  3,  1909,  guinea  pig  2862  received  3  c.  c. 

February  5,  1909,  guinea  pig  28S9  received  24  c.  c. 

February  5,  1909,  guinea  pig  2890  received  2£  c.  c. 

Guinea  pigs  2786  and  2890  died  prematurely  as  a  result  of  the  blood  injections, 
and  the  remaining  8  were  killed  on  the  following  dates  and  on  autopsy  were 
found  to  be  free  from  lesions  of  disease :  Guinea  pigs  2781  and  2782,  killed 
March  27,  1909  (sixty-one  days  after  injection)  ;  guinea  pigs  2783  and  2784, 
killed  April  13,  1909  (seventy-eight  days  after  injection)  ;  guinea  pig  2785, 
killed  April  12,  1909  (seventy-eight  days  after  injection);  guinea  pig  2889, 
killed  April  13,  1909  (sixty-seven  days  after  injection)  ;  guinea  pigs  2861  and 
2862,  killed  April  13,  1909  (sixty-nine  days  after  injection). 

LOT  2. 

Cow  511,  general  condition  poor,  had  been  affected  with  tuberculosis  eighteen 
months  or  more.  Microscopic  examinations  of  the  feces  on  seven  different  days 
revealed  tubercle  bacilli  on  three  days.  A  hog  fed  with  feces  from  the  cow 
contracted  tuberculosis. 

On  February  1,  1909,  two  guinea  pigs,  Nos.  2829  and  2830,  received  each 
an  intraabdominal  injection  of  3  c.  c.  of  blood  from  the  cow.  The  guinea  pigs 
remained  healthy  until  April  13,  1909  (seventy-one  days  after  injection),  when 
they  were  killed  and  found  on  autopsy  to  be  free  from  lesions  of  disease. 

Cow  537,  general  condition  fairly  good,  had  been  affected  with  tuberculosis 
more  than  two  years.  Microscopic  examination  of  the  feces  on  fifteen  days 
revealed  tubercle  bacilli  on  eleven  days.  Guinea  pigs  inoculated  with  small 
masses  of  feces  contracted  tuberculosis. 

Guinea  pigs  were  injected  intraabdominally  with  blood  from  the  cow  as 
follows : 

February  4,  1909,  guinea  pig  2871  received  3  c.  c. 
February  4,  1909,  guinea  pig  2872  received  3  c.  c. 
February  19.  1909,  guinea  pig  3062  received  3  c.  c. 
February  19,  1909,  guinea  pig  3063  received  3  c.  c. 

Guinea  pig  2872  died  prematurely  as  a  result  of  the  injection.  Guinea  pig 
2871  was  killed  April  13,  1909  (sixty-eight  days  after  injection),  and  on  autopsy 
was  found  to  be  free  from  lesions  of  disease.  Guinea  pigs  3062  and  3063  were 
killed  April  13,  1909  (fifty-three  days  after  injection),  and  on  autopsy  were 
found  to  be  free  from  lesions  of  disease. 

Cow  538,  general  condition  very  poor,  had  been  affected  with  tuberculosis 
two  years  or  longer.  Microscopic  examinations  of  feces  on  eleven  different 
days  revealed  tubercle  bacilli  on  nine  days.  Guinea  pigs  inoculated  with 
small  masses  of  feces  and  hogs  fed  feces  of  this  cow  contracted  tuberculosis. 

On  February  3,  1909,  two  guinea  pigs,  Nos.  2851  and  2852,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  blood  from  the  cow.  On  April  13,  1909 
(sixty-nine  days  after  injection),  the  guinea  pigs  were  killed  and  on  autopsy 
were  found  to  be  free  from  lesions  of  disease. 


RESULTS   OF   GUINEA-PIG   INOCULATIONS.  13 

Cow  555,  general  condition  fairly  good,  had  been  affected  with  tuberculosis 
more  than  two  years.  Microscopic  examinations  of  feces  on  five  different  days 
revealed  tubercle  bacilli  on  two  days.  A  hog  fed  feces  from  the  cow  contracted 
tuberculosis. 

On  January  30,  1909,  two  guinea  pigs,  Nos.  2811  and  2812,  received  each  an 
iutraabdominal  injection  of  3  c.  c.  of  blood  from  the  cow.  Guinea  pig  2811 
died  prematurely  as  a  result  of  the  injection.  Guinea  pig  2812  was  killed 
April  12,  1909  (seventy-two  days  after  injection),  and  on  post-mortem  exami- 
nation was  found  to  be  free  from  lesions  of  disease. 

Cow  5G7,  general  condition  good,  had  been  affected  with  tuberculosis  at  least 
two  and  one-half  years.  Microscopic  examinations  of  feces  on  ten  different 
days  revealed  tubercle  bacilli  on  five  days.  A  hog  fed  with  feces  from  the 
cow  contracted  tuberculosis. 

On  February  4,  1909,  two  guinea  pigs,  Nos.  2SG9  and  2870,  received  each  an 
iutraabdominal  injection  of  3  c.  c.  of  blood  from  the  cow.  The  guinea  pigs 
were  killed  April  12,  1909  (sixty-seven  days  after  injection),  and  on  autopsy 
were  found  to  be  free  from  lesions  of  disease. 

Cow  64G,  general  condition  fair;  had  been  affected  with  tuberculosis  for  some 
time,  but  just  how  long  was  not  known.  Microscopic  examinations  of  feces  on 
two  different  days  revealed  tubercle  bacilli  on  one  day.  Guinea  pigs  inoculated 
with  her  feces  contracted  tuberculosis. 

On  February  2,  1909,  two  guinea  pigs,  Xos.  2847  and  2848,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  blood  from  the  cow.  The  guinea  pigs 
were  killed  April  13,  1909  (seventy  days  after  injection),  and  on  autopsy  were 
found  to  be  free  from  lesions  of  disease. 


Cow  503,  general  condition  good;  had  been  affected  with  tuberculosis  at  least 
two  and  one-half  years.  Microscopic  examinations  of  feces  on  two  days  revealed 
tubercle  bacilli  on  one  day. 

On  January  30,  1909,  two  guinea  pigs,  Nos.  2805  and  280G,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  blood  from  the  cow.  The  guinea  pigs 
were  killed  April  30,  1909  (seventy-three  days  after  injection),  and  on  autopsy 
were  found  to  be  free  from  lesions  of  disease. 

Cow  510,  general  condition  fairly  good ;  had  been  affected  with  tuberculosis 
fibout  three  years.  Microscopic  examinations  of  feces  on  three  days  revealed 
tubercle  bacilli  on  two  days. 

Guinea  pigs  were  injected  intraabdominally  with  blood  from  the  cow  as 
follows : 

January  29,  1909,  guinea  pig  2791,  received  5  c.  c. 
January  29,  1909,  guinea  pig  2792,  received  5  c.  c. 
February  4,  1909,  guinea  pig  2SS1,  received  3  c.  c. 
February  4,  1909,  guinea  pig  2882,  received  3  c.  c. 

Guinea  pigs  2791  and  2792  died  prematurely  as  a  result  of  Iho  injections. 
Guinea  pigs  2881  and  2SS2  were  killed  April  13,  1909  (sixty-eight  clays  after 
injection),  and  on  autopsy  were  found  to  be  free  from  lesions  of  disease. 

Cow  512,  general  condition  good;  had  been  affected  with  tuberculosis  eighteen 
months  or  longer.  Microscopic  examinations  of  feces  on  six  different  days 
revealed  tubercle  bacilli  on  four  days. 

On  February  11.  1909.  two  guinea  pigs.  Nos.  2823  and  2X24,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  blood  from  the  cow.  The  guinea  pigs 
were  killed  April  12,  1909  (seventy  days  after  injection),  and  on  autopsy  were 
found  to  be  free  from  lesions  of  disease. 


14  TESTS   CONCERNING   TUBERCLE   BACILLI   IN    THE   BLOOD. 

Cow  513,  general  condition  fairly  good,  but  liad  greatly  enlarged  throat 
glands ;  bad  been  affected  witb  tuberculosis  eighteen  months  or  longer.  Micro- 
scopic examinations  of  feces  on  four  different  days  revealed  tubercle  bacilli  on 
two  days. 

On  February  2,  1909,  two  guinea  pigs,  Nos.  2835  and  2836,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  blood  from  the  cow.  Guinea  pig  2835  died 
prematurely  as  a  result  of  the  injection.  Guinea  pig  2830  was  killed  April  12, 
1909  (sixty-nine  days  after  injection),  and  on  autopsy  was  found  to  be  free 
from  lesions  of  disease. 

Cow  514,  general  condition  poor ;  had  been  affected  with  tuberculosis  about 
three  years.  Microscopic  examinations  of  feces  on  three  different  days  revealed 
tubercle  bacilli  on  one  day. 

On  January  30,  1909,  two  guinea  pigs,  Nos.  2815  and  2S16,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  blood  from  the  cow.  Guinea  pig  281G  died 
of  an  intercurrent  affection  March  3,  1909  (thirty-two  days  after  injection),  and 
on  autopsy  was  found  to  be  free  from  lesions  of  tuberculosis.  Guinea  pig  2815 
was  killed  April  12,  1909  (seventy-two  days  after  injection),  and  on  autopsy 
was  found  to  be  free  from  lesions  of  disease.  4 

Cow  515,  general  condition  fair,  had  been  affected  with  tuberculosis  eighteen 
months  or  longer.  Microscopic  examinations  of  feces  on  six  different  days 
revealed  tubercle  bacilli  on  four  days. 

On  February  1,  1909,  two  guinea  pigs,  Nos.  2821  and  2822,  received  each  an 
intraabdoniiual  injection  of  3  c.  c.  of  blood  from  the  cow.  The  guinea  pigs  were 
killed  April  12,  1909  (seventy  days  after  injection),  and  on  autopsy  were  found 
to  be  free  from  lesions  of  disease. 

Cow  516,  general  condition  fairly  good,  had  been  affected  with  tuberculosis 
eighteen  months  or  longer.  Microscopic  examinations  of  feces  on  seven  different 
days  revealed  tubercle  bacilli  on  four  days. 

On  February  1,  1909,  two  guinea  pigs,  Nos.  2831  and  2832,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  blood  from  the  cow.  The  guinea  pigs 
were  killed  April  13,  1909  (seventy-one  days  after  injection),  and  on  autopsy 
were  found  to  be  free  from  lesions  of  disease. 

Cow  536,  general  condition  poor,  had  been  affected  with  tuberculosis  two  years 
or  longer.  Microscopic  examinations  of  feces  on  eleven  different  days  revealed 
tubercle  bacilli  on  eight  days. 

On  February  3,  1909,  two  guinea  pigs,  Nos.  2865  and  2866,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  blood  from  the  cow.  The  guinea  pigs 
were  killed  April  13,  1909  (sixty -nine  days  after  injection),  and  on  autopsy 
were  found  to  be  free  from  lesions  of  disease. 

Cow  551,  general  condition  fairly  good,  had  been  affected  with  tuberculosis 
two  years  or  longer.  Microscopic  examinations  of  feces  on  nine  different  days 
revealed  tubercle  bacilli  on  three  days. 

On  February  1,  1909,  two  guinea  pigs,  Nos.  2833  and  2834,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  blood  from  the  cow.  The  guinea  pigs  were 
killed  April  12,  1909  (seventy  days  after  injection),  and  on  autopsy  were  found 
to  be  free  from  lesions  of  disease. 

Cow  553,  general  condition  fairly  good,  had  been  affected  with  tuberculosis 
two  years  or  longer.  Microscopic  examinations  of  feces  on  three  different  days 
revealed  tubercle  bacilli  every  day. 

On  February  1,  1909,  two  guinea  pigs,  Nos.  2827  and  2828,  received  each  an 
iutraabdominal  injection  of  3  c.  c.  of  blood  from  the  cow.  The  guinea  pigs 
were  killed  April  13,  1909  (seventy-one  days  after  injection),  and  on  autopsy 
were  found  to  be  free  from  lesions  of  disease. 


RESULTS   OF   GUINEA-PIG  INOCULATIONS.  15 

Cow  G20,  general  condition  good,  had  been  affected  with  tuberculosis  a  year 
or  longer.  Microscopic  examinations  of  feces  on  four  different  days  revealed 
tubercle  bacilli  on  one  day. 

On  February  3,  1009,  two  guinea  pigs,  Nos.  2855  and  285G,  received  each  an 
intraabdoniinal  injection  of  3  c.  c.  of  blood  from  the  cow.  The  guinea  pigs 
were  killed  April  13,  1909  (sixty-nine  days  after  injection),  and  on  autopsy 
were  found  to  be  free  from  lesions  of  disease. 

Cow  629,  general  condition  fair,  had  been  affected  with  tuberculosis  at  least 
one  year.  Microscopic  examinations  of  feces  on  five  different  days  revealed 
tubercle  bacilli  on  two  days. 

On  February  3,  1909,  two  guinea  pigs.  Nos.  2857  and  2858,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  blood  from  the  cow.  The  guinea  pigs 
were  killed  April  13,  1909  (sixty-nine  days  after  injection),  and  on  autopsy 
were  found  to  be  free  from  lesions  of  disease. 

Cow  631,  general  condition  fair,  had  been  affected  with  tuberculosis  at  least 
one  year.  Microscopic  examinations  of  feces  on  four  different  days  revealed 
tubercle  bacilli  on  one  day.  ' 

On  February  4,  1909,  two  guinea  pigs,  Nos.  2877  and  2878,  received  each 
an  intraabdominal  injection  of  3  c.  c.  of  blood  from  the  cow.  The  guinea  pigs 
were  killed  April  13,  1909  (sixty-eight  days  after  injection),  and  on  autopsy 
were  found  to  be  free  from  lesions  of  disease. 

Bull  635,  general  condition  good,  had  been  affected  with  tuberculosis  over 
two  years.  Microscopic  examinations  of  feces  on  three  different  days  revealed 
tubercle  bacilli  on  one  day. 

On  January  20,  1909.  two  guinea  pigs,  Nos.  2817  and  2818,  received  each  an 
intraabdominal  injection  of  3  .c.  c.  of  blood  from  the  bull.  Guinea  pig  2818 
died  prematurely  as  a  result  of  the  injection.  Guinea  pig  2817  was  killed  April 
12,  1909  (seventy-two  days  after  injection),  and  on  autopsy  was  found  to  be 
free  from  lesions  of  disease. 

Cow  636,  general  condition  fairly  good,  had  been  affected  with  tuberculosis 
at  least  one  year.  Microscopic  examinations  of  feces  on  five  different  days 
revealed  tubercle  bacilli  on  two  days. 

On  January  30,  1909,  two  guinea  pigs,  Nos.  2803  and  2804,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  blood  from  the  cow.  The  guinea  pigs 
were  killed  April  12,  1909  (seventy-two  days  after  injection),  and  on  autopsy 
were  found  to  be  free  from  lesions  of  disease. 

Cow  638,  general  condition  fairly  good,  had  been  affected  with  tuberculosis 
over  two  years.  Microscopic  examinations  of  feces  on  four  different  days 
revealed  tubercle  bacilli  on  all  four  days. 

Guinea  pigs  were  given  intraabdominal  injections  of  the  blood  of  this  cow 
as  follows  : 

January  29,  1909,  guinea  pig  2795  received  5  c.  c. 
January  29.  1909,  guinea  pig  2796  received  5  c.  c. 
February  4,  1909,  guinea  pig  2879,  received  3  c.  c. 
February  4,  1909.  guinea  pig  2880  received  3  c.  c. 

Guinea  pig  2795  died  prematurely  as  a  result  of  the  injection.  Guinea  pig 
2796  was  killed  April  12,  1909  (seventy-three  days  after  the  injection),  and  on 
autopsy  was  found  to  be  free  from  lesions  of  disease.  Guinea  pigs  2879  and 
2880  were  killed  April  12,  1909  (sixty-seven  days  after  injection),  and  on 
autopsy  were  found  to  be  free  from  lesions  of  disease. 

Cow  639,  general  condition  good,  had  been  affected  with  tuberculosis  at  least 
one  year.  Microscopic  examinations  of  feces  on  four  different  days  revealed 
tubercle  bacilli  oil  one  day. 


16  TESTS   CONCERNING  TUBERCLE   BACILLI   IN   THE   BLOOD. 

Guinea  pigs  were  given  intraabdominal  injections  of  the  blood  of  this  cow 
as  follows : 

January  29,  1900,  guinea  pig  2801  received  5  c.  c. 
January  29,  1909,  guinea  pig  2802  received  5  c.  c. 
February  5,  1909,  guinea  pig  2887  received  3  c.  c. 
February  5,  1909,  guinea  pig  2888  received  3  c.  c. 

Guinea  pig  2S01  died  prematurely  as  a  result  of  the  injection.  Guinea  pig 
2802  was  killed  April  12,  1909  (seventy-three  days  after  injection),  and  on 
autopsy  several  small  necrotic  foci  were  found  in  the  liver  and  spleen.  The 
lesions  were  not  at  all  like  the  conditions  caused  by  the  tubercle  bacillus, 
and  microscopic  examinations  failed  to  reveal  tubercle  bacilli.  Some  of  the 
abnormal  tissue  was  used  to  make  subinoculations  into  guinea  pigs ;  the  sub- 
inoculated  guinea  pigs  failed  to  show  tuberculosis. 

Guinea  pigs  2887  and  2888  were  killed  April  13,  1909  (sixty-seven  days  after 
the  injection),  and  on  autopsy  were  found  to  be  free  from  lesions  of  disease. 

Cow  640,  general  condition  good,  had  been  affected  with  tuberculosis  over 
two  years.  Microscopic  examinations  of  feces  on  three  different  days  revealed 
tubercle  bacilli  on  all  three  days. 

On  January  29,  1909,  two  guinea  pigs,  Nos.  2799  and  2800,  received  each  an 
intraabdominal  injection  of  5  c.  c.  of  blood  from  the  cow.  The  guinea  pigs 
were  killed  April  12  1909  (seventy-three  days  after  injection),  and  on  autopsy 
were  found  to  be  free  from  lesions  of  disease. 

Cow  642,  general  condition  good,  had  been  affected  with  tuberculosis  at  least 
one  year.  Microscopic  examinations  of  feces  on  three  different  days  revealed 
tubercle  bacilli  on  two  days. 

Guinea  pigs  were  injected  intraabdominally  with  blood  from  this  cow  as 
follows : 

January  29,  1909,  guinea  pig  2793  received  5  c.  c. 
January  29,  1909,  guinea  pig  2794  received  5  c.  c. 
February  5,  1909,  guinea  pig  2885  received  3  c.  c. 
February  5,  1909,  guinea  pig  2886  received  3  c.  c. 

Guinea  pig  2794  died  prematurely  as  the  result  of  the  injection.  Guinea  pig 
2886  died  March  5,  1909  (twenty-eight  days  after  injection),  of  an  intercurrent 
affection,  and  the  autopsy  revealed  no  lesions  resembling  tuberculosis.  Guinea 
pig  2793  was  killed  April  13,  1909  (seventy-three  days  after  injection),  and  on 
autopsy  was  found  to  be  free  from  lesions  of  disease.  Guinea  pig  2885  was 
killed  April  13,  1909  (sixty-seven  days  after  injection),  and  on  autopsy  was 
found  to  be  free  from  lesions  of  disease. 

LOT  4. 

Cow  479,  general  condition  fair,  but  had  greatly  enlarged  throat  glands,  had 
been  affected  with  tuberculosis  about  three  years. 

On  February  4,  1909,  two  guinea  pigs,  Nos.  2873  and  2874,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  her  blood.  The  guinea  pigs  were  killed 
April  13,  1909  (sixty-eight  days  after  injection),  and  on  autopsy  were  found  to 
be  free  from  lesions  of  disease. 

Bull  508,  general  condition  good,  had  been  affected  with  tuberculosis  about 
two  and  one-half  years. 

On  February  4,  1909,  two  guinea  pigs,  Nos.  2867  and  2868,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  his  blood.  The  guinea  pigs  were  killed 
April  12,  1909  (sixty-seven  days  after  injection),  and  on  autopsy  were  found 
to  be  free  from  lesions  of  disease. 

Cow  517,  general  condition  good,  had  been  affected  with  tuberculosis  about 
two  and  one-half  years. 


RESULTS   OF   GUINEA-PIG   INOCULATIONS.  17 

On  February  1,  190!),  two  guinea  pigs,  Nos.  2825  and  2820,  received  each  an 
intraabdoiuinal  injection  of  3  c.  c.  of  her  blood.  Guinea  pig  2825  died  of  an 
Intel-current  affection  April  5,  1909  (sixty-three  days  after  injection),  and  on 
autopsy  was  found  to  be  free  from  lesions  of  tuberculosis.  Guinea  pig  2826 
was  killed  April  13,  1909  (seventy-one  days  after  injection),  and  on  autopsy 
was  found  to  be  free  from  lesions  of  disease. 

Cow  569,  general  condition  fair,  had  been  affected  with  tuberculosis  about 
two  years. 

On  February  1,  1909,  two  guinea  pigs,  Nos.  2819  and  2820,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  her  blood.  The  guinea  pigs  were  killed 
April  12.  1909  (seventy  days  after  injection),  and  on  autopsy  were  found  to  be 
free  from  lesions  of  disease. 

Cow  630,  general  condition  fair,  had  been  affected  with  tuberculosis  about 
one  year. 

On  February  3,  1909,  two  guinea  pigs,  Nos.  2853  and  2854,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  her  blood.  The  guinea  pigs  were  killed 
April  13,  1909  (sixty -nine  days  after  injection),  and  on  autopsy  were  found  to 
be  free  from  lesions  of  disease. 

Cow  632,  general  condition  good,  had  been  affected  with  tuberculosis  at  least 
one  year. 

On  January  30,  1909,  two  guinea  pigs,  Nos.  2807  and  2808,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  her  blood.  Guinea  pig  2808  died  prema- 
turely as  a  result  of  the  injection.  Guinea  pig  2807  was  killed  April  13,  1909 
(seventy-two  days  after  injection),  and  on  autopsy  was  found  to  be  free  from 
lesions  of  disease. 

Cow  633,  general  condition  fair,  had  been  affected  with  tuberculosis  at  least 
one  year. 

On  January  30,  1909,  two  guinea  pigs.  Nos.  2813  and  2814,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  her  blood.  Guinea  pig  2813  died  prema- 
turely as  a  result  of  the  injection.  Guinea  pig  2814  was  killed  on  April  12, 
1909  (seventy-one  days  after  injection),  and  on  autopsy  was  found  to  be  free 
from  lesions  of  disease. 

Cow  634,  general  condition  fair,  had  been  affected  with  tuberculosis  at  least 
one  year. 

On  January  29,  1909.  two  guinea  pigs,  Nos.  2787  and  2788,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  her  blood.  The  guinea  pigs  were  killed 
April  12.  1909  (seventy-three  days  after  injection),  and  on  autopsy  were  found 
to  be  free  from  lesions  of  disease. 

Cow  641.  general  condition  fair,  had  been  affected  with  tuberculosis  over 
two  years. 

On  January  29,  1909.  two  guinea  pigs.  Nos.  2797  and  2798.  received  each  an 
intraabdominal  injection  of  5  c.  c.  of  the  blood  of  the  cow.  and  on  February 
4.  1909.  two  guinea  pigs.  Nos.  2875  and  2876.  received  each  a  similar  injection 
of  3  c.  c.  of  blood.  Guinea  pig  2798  died  prematurely  as  a  result  of  the  injec- 
tion. Guinea  pig  2797  was  killed  April  12.  1909  (seventy-five  days  after  in- 
jection), and  guinea  pigs  2875  and  2876  were  killed  April  12.  1909  (sixty- 
seven  days  after  injection).  The  last  three  guinea  pigs  were  found  to  be  free 
from  lesions  of  disease. 

Cow  644.  general  condition  fair,  had  been  affected  with  tuberculosis  four 
months  or  more. 

On  February  2.  1909.  two  guinea  pigs.  Nos.  2849  and  2x50.  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  blood  of  the  cow.  The  guinea  pi;;*  were 
killed  April  13,  190!)  (seventy  days  after  injection),  and  on  autopsy  were  found 
to  be  free  from  lesions  of  disease. 


18  TESTS   CONCERNING  TUBERCLE   BACILLI   IN    THE   BLOOD. 

Cow  645,  general  condition  good,  had  been  affected  with  tuberculosis  three 
mouths  or  more. 

On  February  2,  1909,  two  guinea  pigs,  Nos.  2845  and  2846,  received  each  an 
iutraabdoniinal  injection  of  3  c.  c.  of  her  blood.  Guinea  pig  2845  died  pre- 
maturely as  a  result  of  the  injection.  Guinea  pig  2846  was  killed  April  13, 
1909  (seventy  days  after  injection),  and  on  autopsy  was  found  to  be  free  from 
lesions  of  disease. 

Cow  648,  general  condition  good,  had  been  affected  with  tuberculosis  for  an 
unknown  period  of  time.  She  was  brought  to  the  Experiment  Station  shortly 
before  her  blood  was  used  for  guinea-pig  injections,  and  reacted  with  tuber- 
culin. 

On  February  2,  1909,  two  guinea  pigs,  Nos.  2837  and  2838,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  her  blood.  The  guinea  pigs  were  killed 
April  12,  1909  (sixty-nine  days  after  injection),  and  on  autopsy  were  found 
to  be  free  from  lesions  of  disease. 

Cow  657,  general  condition  fair,  had  been  affected  with  tuberculosis  for  an 
unknown  period  of  time.  She  was  brought  to  the  Experiment  Station  shortly 
before  her  blood  was  used  for  guinea-pig  injections,  and  reacted  with  tuber- 
culin. 

On  February  2,  1909,  two  guinea  pigs,  Nos.  2841  and  2842,  received  each  an 
intraabdominal  injection  of  3  c.  c.  of  her  blood.  The  guinea  pigs  were  killed 
April  12,  1909  (sixty-nine  days  after  injection),  and  on  autopsy  were  found 
to  be  free  from  lesions  of  disease. 

DISCUSSION   OF   RESULTS. 

Among  the  42  cattle  enumerated  above,  27,  or  64|  per  cent,  were 
shown  by  microscopic  examinations  to  be  discharging  tubercle  bacilli 
from  their  bowels — in  most  instances  intermittently — and  the  infec- 
tious character  of  the  feces  in  7  cases,  or  16§  per  cent,  was  demon- 
strated by  animal  experiments — that  is,  feeding  and  inoculation  tests. 

These  two  facts — that  27  of  the  cattle  were  shown  by  microscopic 
tests  to  be  expelling  tubercle  bacilli  per  rectum,  Avhile  only  7  were 
proved  by  animal  experiments  to  be  passing  infected  feces — must 
not  be  taken  as  being  in  any  sense  contradictory,  as  the  feces  of  only 
a  sufficient  number  of  tuberculous  cattle  were  tested  by  animal  feeding 
and  inoculation  experiments  to  prove  conclusively  that  the  acid-fast 
bacilli  found  on  microscopic  examinations  in  the  feces  of  tuberculous 
cattle  are  certainly  live,  virulent  tubercle  bacilli. 

Relative  to  the  expulsion  of  tubercle  bacilli  from  the  bowels  of 
tuberculous  cattle,  all  the  evidence  we  have  indicates  that  the  bacilli 
have  their  origin  in  the  lung  and  throat,  from  which  regions  they 
are  coughed  up,  swallowed,  and  passed  through  and  out  of  the  intes- 
tinal canal  without  appreciable  loss  of  pathogenic  virulence.  That  a 
large  proportion  of  the  tubercle  bacilli  swallowed  by  cattle  really 
pass  through  their  bodies  and  out  per  rectum  without  a  determinable 
loss  of  virulence  was  experimentally  shown  in  some  of  our  earlier 
work."  We  have  absolutely  no  reason  to  believe  that  tubercle  bacilli 

a  Bureau  of  Animal  Industry,  Bulletins  SS  and  99. 


DISCUSSION    OF   RESULTS.  19 

enter  the  intestinal  canal  from  the  lymph  radicals  or  blood  capil- 
laries or  by  any  complex  and  mysterious  system  of  transporta- 
tion from  lesions  of  all  descriptions  and  kinds  in  any  or  every 
portion  of  the  body.  It  is  our  conviction  that,  unless  an  open  tuber- 
culosis is  in  more  or  less  direct  communication  with  the  intestinal 
canal  or  there  is  a  tuberculous  disease  of  the  intestine  itself,  which 
latter  is  rare  among  cattle,  no  tubercle  bacilli  will  be  expelled  with 
the  feces. 

If  tuberculosis  in  all  its  forms  was  a  bacteriemia  the  expulsion  of 
tubercle  bacilli  from  the  bowels  of  all  tuberculous  individuals,  as  well 
as  with  their  urine,  saliva,  milk,  and  other  bodily  secretions,  would 
follow  as  a  natural  consequence.  Those  who  have  carefully  studied 
the  secretions  from  the  uninvolved  organs  of  tuberculous  subjects 
know  how  rarely  tubercle  bacilli  are  detected  in  them  even  with  the 
application  of  the  most  delicate  tests. 

When  we  consider  cattle  like  Xos.  533,  549,  and  552 — three  of 
the  four  animals  of  which  autopsy  records  are  given — and  note  that 
they  were  so  badly  diseased  that  they  would  have  been  condemned  on 
superficial  examination  as  wholly  unfit  for  use  as  food  under  the 
existing  meat-inspection  regulations,  the  absence  of  tubercle  bacilli 
from  their  blood  may  be  regarded  as  a  sufficient  reason  for  assuming 
that  the  possible  occurrence  of  tubercle  bacilli  in  the  blood  of  tuber- 
culous animals  will  almost  invariably  be  associated  with  pathological 
conditions  of  a  very  marked  character,  or  that  the  tubercle  bacilli  will 
be  present  in  extremely  small  numbers  and  will  speedily  be  filtered 
out  of  the  blood  stream.  Cow  533  had  been  affected  with  tuberculosis 
two  years  or  longer,  was  in  poor  condition  as  a  result  of  the  disease, 
and  on  autopsy  was  found  to  have  an  extensive,  open  tuberculosis  of 
the  lung  and  lesions  of  tuberculosis  in  the  liver  and  in  both  the 
thoracic  and  abdominal  lymph  glands.  Cow  549  was,  if  anything, 
even  more  severely  and  extensively  affected,  and  had  given  birth  to  a 
congenitally  tuberculous  calf  less  than  a  year  before  her  blood  was 
injected  into  guinea  pigs.  Cow  552  was  also  affected  with  general- 
ized, advanced,  open  tuberculosis,  and  prior  to  the  use  of  her  blood 
for  the  guinea-pig  injections  was  found  to  be  passing  from  her  bowels 
large  numbers  of  tubercle  bacilli,  which  were  proved  by  feeding  tests 
to  be  virulent  for  hogs  and  by  inoculation  tests  to  be  virulent  for 
guinea  pigs.  With  the  blood  obtained  from  these  three  cows  14 
guinea  pigs  were  injected,  of  which  2  died  prematurely  and  12  lived 
two  months  or  more  afterwards,  until  they  were  intentionally  killed, 
when  they  were  found  on  post-morten  examination  to  be  wholly  free 
from  lesions  of  disease  of  any  kind. 

The  possibility  exists  that  tubercle  bacilli  introduced  into  the 
stomach  and  intestine  by  swallowing  may  be  taken  up  by  the  lymph 
radicals,  passed  along  the  lymph  channels,  and  emptied  through  the 


20  TESTS   CONCERNING   TUBERCLE    BACILLI    IN    THE   BLOOD. 

great  lymph  ducts  into  the  venous  circulation.  The  investigations 
of  Nicolas  and  Descos,  Ravenel,  Calmette  and  Guerin,  Schlossman 
and  Engle,  and  others  speak  for  this;  but  such  tubercle  bacilli  will 
not  be  very  numerous  and  will  no  doubt  be  filtered  out  of  the  blood 
as  soon  as  it  reaches  the  lung  through  the  heart  and  pulmonary 
arteries,  to  which  it  passes  directly  after  it  has  received  the  lymph 
stream. 

SUPPLEMENTAL   TESTS  REGARDING  POSSIBLE  IMMUNITY. 

We  have  already  stated  that  the  possibility  exists  that  the  intra- 
peritoneal  injection  of  from  3  to  5  c.  c.  of  fresh,  warm  blood  from 
tuberculous  cattle  induces  an  immunity  in  guinea  pigs  to  the  tubercle 
bacilli  the  blood  may  contain.  Although  we  knew  of  nothing  to 
uphold  this  theory,  we  considered  it  necessary  to  undertake  an  investi- 
gation to  prove  or  disprove  it,  the  results  of  which  are  now  presented. 

On  April  24,  1909,  blood  and  tuberculous  material  was  obtained 
from  cow  533  (see  record  of  cow  on  p.  11)  for  a  number  of  guinea- 
pig  injections.  The  primary  object  of  the  injections  Was  to  prove 
that  the  blood  of  a  tuberculous  cow,  when  introduced  into  the  peri- 
toneal cavity  of  a  guinea  pig,  has  no  retarding  influence  on  the  devel- 
opment of  tuberculosis  from  tubercle  bacilli  that  may  be  present  in  it. 

Cow  533  was  first  bled  from  the  jugular  vein  and  then  at  once 
killed.  As  soon  as  she  was  dead  a  tuberculous  mediastinal  gland  was 
removed  from  her  body  and  500  mg.  of  it  emulsified  with  2  c.  c.  of 
sterile,  normal  salt  solution.  Cover  glasses  of  this  emulsion,  stained 
with  carbolfuchsin  and  decolorized  with  20  per  cent  sulphuric  acid, 
revealed  on  microscopic  examination,  on  an  average,  two  tubercle 
bacilli  each.  The  emulsion  was  mixed  with  an  additional  quantity  of 
sterile,  normal  salt  solution,  so  that  each  cubic  centimeter  of  the 
dilution  represented  a  strength  equal  to  one  drop  of  the  original 
emulsion. 

The  blood  obtained  from  the  cow  prior  to  her  death  and  the  diluted 
emulsion  made  with  the  tuberculous  mediastinal  gland  from  her  body 
were  used  to  inject  seven  groups  of  guinea  pigs,  the  records  of  which 
are  given  below. 

The  guinea  pigs  in  the  seven  different  groups  were  injected  for  the 
following  purposes:  Group  1,  to  serve  as  checks  on  the  absence  or 
presence  of  tubercle  bacilli  in  the  blood  of  the  tuberculous  cow  that 
was  used  for  the  investigation ;  group  2,  to  show  that  the  intra- 
abdominal  injection  of  fresh,  warm  blood  from  a  tuberculous  cow 
can  not  protect  against  tubercle  bacilli  simultaneously  introduced 
into  the  abdominal  cavity ;  group  3,  to  show  that  the  intraabdominal 
injection  of  fresh,  warm  blood  from  a  tuberculous  cow  can  not  pro- 
tect against  tubercle  bacilli  introduced  into  other  parts  of  the  body 
than  the  abdominal  cavity ;  groups  4  and  5,  to  show  that  the  blood  of 


SUPPLEMENTAL   TESTS   REGARDING   IMMUNITY.  21 

tuberculous  cows  has  no  special  germicidal  potency  for  tubercle 
bacilli ;  groups  G  and  7,  to  serve  as  guides  relative  to  the  amount  of 
tuberculous  disease  to  be  expected  in  the  bodies  of  the  guinea  pigs 
that  were  injected  with  both  blood  and  emulsion  of  tuberculous 
material. 

GROUP  1. 

On  April  24,  1909,  eight  guinea  pigs,  Nos.  3626  to  3633,  inclusive,  received 
each  an  intraahdouiinal  injection  of  3  c.  c.  of  freshly  drawn  warm  blood.  On 
May  6,  1909.  guinea  pig  3(527  died  affected  with  congestion  of  the  lungs.  On 
autopsy  no  lesions  of  tuberculosis  were  found.  On  May  27  and  28,  1909,  guinea 
pigs  3626,  3628,  3629,  3630,  3(531,  3632.  and  3633  were  killed  and  examined  post- 
mortem. No  lesions  of  tuberculosis  or  other  disease  were  found. 

GROUP  2. 

On  April  24,  1909,  eight  guinea  pigs,  Nos.  3642  to  3649,  inclusive,  received 
each  an  intraabdominal  injection  of  3  c.  c.  of  freshly  drawn  blood,  followed 
as  quickly  as  possible  by  an  intraabdominal  injection  of  0.5  c.  c.  of  tuberculous 
emulsion.  On  April  30,  1909,  guinea  pig  3642  died  affected  with  inflammation 
of  the  large  bowel.  On  May  27,  1909.  guinea  pigs  3643,  3644,  3645,  3(546,  3647, 
3648,  and  3649  were  killed  and  examined  post-mortem.  Every  one  of  the  seven 
was  found  to  be  affected  with  generalized  tuberculosis  of  the  abdominal  and 
thoracic  organs. 

GROUP  :5. 

On  April  24,  1909,  eight  guinea  pigs,  Nos.  3634  to  3641,  inclusive,  received  each 
an  intraabdominal  injection  of  freshly  drawn  warm  blood,  followed  as  soon  as 
possible  by  a  subcutaneous  injection  into  the  right  thigh  of  0.5  c.  c.  of  tuber- 
culous emulsion.  On  May  27,  1909,  guinea  pigs  3636  and  3637  and  on  May  28, 
1909,  guinea  pigs  3634,  3635,  3638,  3639,  3640,  and  3641  were  killed  and  examined 
post-mortem.  The  eight  guinea  pigs  all  showed  more  or  less  extensive  lesions 
of  tuberculosis  at  the  seat  of  the  subcutaneous  injection,  tuberculosis  of  the 
adjacent  superficial  inguinal  gland,  tuberculosis  of  the  pelvic,  lumbar,  and 
gastro-hepatic  glands,  and  a  sprinkling  of  tuberculous  foci  in  the  liver  and 
spleen. 

GROUP  4. 

On  April  24,  1909,  eight  guinea  pigs,  Nos.  3658  to  3665,  inclusive,  received  each 
an  intraabdominal  injection  of  3  c.  c.  of  a  mixture  of  defibrinated  blood  and 
tuberculous  emulsion.  Each  3  c.  c.  of  this  mixture  was  equivalent  to  0.5  c.  c 
of  the  diluted  tuberculous  emulsion  described  earlier.  The  mixture  was  two 
hours  old  at  the  time  it  was  injected  into  the  guinea  pigs.  On  April  25,  1909, 
guinea  pigs  3659,  3660,  and  3661  died.  The  post-mortem  examinations  showed 
no  lesions  excepting  a  congested  condition  of  the  lungs  and  a  quantity  of 
unabsorbed  blood  in  the  peritoneal  cavity.  On  May  12,  1909,  guinea  pig  3662 
died  affected  with  congestion  of  the  lungs;  no  lesions  of  tuberculosis  were  found. 
On  May  27,  1909,  guinea  pigs  3658,  3663,  3664,  and  3665  were  killed  and  exam- 
ined post-mortem.  Three  of  them  were  affected  with  completely  generalized 
tuberculosis  of  the  abdominal  and  thoracic  organs,  and  the  remaining  one  (No. 
3665)  with  generalized  tuberculosis  of  the  abdominal  organs  only. 

GROUP   ">. 

On  April  26,  1909,  eight  guinea  pigs,  Nos.  3670  to  3(577,  inclusive,  received 
each  an  intraabdominal  injection  of  3  c.  c.  of  the  same  mixture  of  blood  and 


22  TESTS   CONCERNING   TUBERCLE   BACILLI   IN   THE   BLOOD. 

tuberculous  emulsion  used  for  tlie  guinea  pigs  of  group  4.  The  mixture  was 
forty-five  hours  old  at  the  time  it  was  injected.  On  May  27,  1909,  the  eight 
guinea  pigs  were  killed  and  examined  post-mortem.  Seven  of  them  were 
affected  with  generalized  tuberculosis  of  the  abdominal  and  thoracic  organs, 
and  one  (No.  3672)  with  generalized  tuberculosis  of  the  abdominal  organs  only. 

GROUP  6. 

On  April  24,  1909,  four  guinea  pigs,  Nos.  3650  to  3653,  inclusive,  received 
each  an  intraabdominal  injection  of  0.5  c.  c.  of  tuberculous  emulsion.  On 
May  6,  1909,  guinea  pig  3651  died  affected  with  congestion  of  the  lungs.  On 
May  27,  1909,  Nos.  3650,  3652,  and  3653  were  killed  and  examined  post-mortem. 
No.  3650  showed  tuberculous  lesions  of  the  spleen  and  omentum  only,  and  Nos. 
3652  and  3653  showed  a  fairly  generalized  tuberculosis  of  the  abdominal  and 
Thoracic  organs. 

GROUP  7. 

On  April  24,  1909,  four  guinea  pigs,  Nos  3654  to  3657,  inclusive,  received  each 
a  subcutaneous  injection,  in  the  right  thigh,  of  0.5  c.  c.  of  tuberculous  emulsion. 
On  May  27,  1909,  the  four  guinea  pigs  were  killed  and  examined  post-mortem. 
Nos.  3654  and  3656  each  showed  a  small  tuberculous  abscess  at  the  seat  of 
injection,  a  tuberculous  condition  of  the  superficial  inguinal  gland  near  the 
seat  of  injection,  and  a  fairly  generalized  tuberculosis  of  the  pelvic  and  abdomi- 
nal organs.  Guinea  pigs  3655  and  3657  showed  similar  lesions  with  the  excep- 
tion of  the  abscesses  at  the  seat  of  injection. 

The  autopsy  records  of  the  guinea  pigs  show,  in  a  general  way, 
very  little  difference  between  the  animals  that  received  only  tuber- 
culous emulsion  and  those  that  received  both  blood  and  emulsion. 
The  guinea  pigs  that  received  both  blood  and  emulsion  into  their 
abdominal  cavities  showed  numerically  more  extensive  lesions  of 
tuberculosis  than  the  guinea  pigs  that  received  only  emulsion  into 
their  abdominal  cavities.  This  condition  would  naturally  be  ex- 
pected because  the  same  number  of  tubercle  bacilli  contained  in  3 
c.  c.  of  blood  would  be  mr>re  widely  separated  and  in  better  condi- 
tion to  start  a  large  number  of  individual  Iqsions  than  those  in 
0.5  c.  c.  of  salt  solution. 

The  use  of  an  emulsion  of  tuberculous  tissue  from  the  tuberculous 
cow  that  supplied  the  blood  for  the  supplemental  injections  was 
preferred  to  the  use  of  a  pure  culture  of  tubercle  bacilli,  because  it 
seemed  desirable  to  us  to  use  infectious  material  and  blood  in  this 
instance  from  the  same  individual  case  of  tuberculosis. 

The  total  number  of  guinea  pigs  injected  in  this  supplemental 
investigation  was  48,  of  which  8  received  blood  only,  32  both  blood 
and  tuberculous  material,  and  8  tuberculous  material  only.  Of  the 
32  that  received  both  blood  and  tuberculous  material  and  the  8  that 
received  only  tuberculous  material,  6  died  prematurely,  and  the 
remaining  34,  when  they  were  killed — thirty  to  thirty-one  days  after 
the  injection — were  all  found  to  be  affected  with  tuberculosis  of  a 
form  that  would  have  progressed  to  death  in  a  short  time. 


CONCLUSIONS.  23 

Among  the  8  guinea  pigs  that  received  an  injection  of  fresh  warm 
blood  without  the  addition  of  tuberculous  material,  1  died  prema- 
turely and  the  remaining  7  were  found  on  autopsy  to  be  free  from 
lesions  of  disease.  Since  the  cow  that  supplied  the  blood  for  the 
injections  was  affected,  as  her  record  shows,  with  extensive,  advanced 
tuberculosis,  the  7  guinea  pigs  make  a  strong  addition  to  the  88 
parallel  cases  of  which  the  records  have  been  previously  given;  and 
hence  we  have  95  guinea  pigs  as  the  total  number  that  received  injec- 
tions of  blood  from  tuberculous  cattle  into  their  peritoneal  cavities — 
the  most  delicate  test  for  tubercle  bacilli  available — and  survived  the 
injection  long  enough  for  tuberculosis  to  manifest  itself  clearly. 
Among  this  total  of  95.  guinea  pigs  not  one  case  of  tuberculosis 
developed.0 

CONCLUSIONS. 

1.  We  failed  utterly  to  find  tubercle  bacilli  in  the  blood  of  tuber- 
culous cattle  which  we  examined  microscopically  in  accordance  with 
the  method  described  and  used  by  Doctor  Rosenberger. 

2.  The  negative  results  of  our  microscopic  examinations  are  con- 
firmed by  the  negative  results  obtained  with  95  guinea  pigs,  each  of 
Avhich  received  an  intraabdominal  injection  of  blood  from  a  tuber- 
culous cow  or  bull. 

3.  As  the  number  of  cattle  from  which  blood  was  injected  into  the 
95  guinea  pigs  was  42,  and  as  these  cattle  represented  practically  all 
stages  of  tuberculosis  from  mildly  affected  recent  cases  to  old  and 
completely  generalized  cases,  we  feel  that  our  work  shows  beyond 
the  remotest  doubt  that  tuberculosis  is  not  to  be  classified,  in  any 
sense  of  the  word,  as  a  bacteriemia. 

°An  independent  investigation  relative  to  the  occurrence  of  tubercle  bacilli 
in  the  circulating  blood  of  cattle  was  made  in  the  Bureau  of  Animal  Industry 
by  Dr.  John  R.  Mohler,  chief  of  the  Pathological  Division.  Mohler  examined 
the  blood  of  8  cattle  microscopically,  and  with  blood  from  each  of  these  cattle 
injected  5  guinea  pigs.  The  microscopic  examinations  and  injections  were  made 
precisely  in  the  manner  described  by  Doctor  Rosenberger.  No  tubercle  bacilli 
were  discovered  microscopically,  and  not  one  of  the  40  injected  guinea  pigs  con- 
tracted tuberculosis.  Two  of  the  8  cattle  were  in  good  condition,  but  were 
passing  tubercle  bacilli  from  their  bowels;  2  of  the  cattle  were  in  poor  con- 
dition and  were  passing  tubercle  bacilli  from  their  bowels;  and  4  of  the  cattle 
were  slaughtered  for  meat,  but  on  inspection  were  found  to  be  so  extensively 
affected  with  tuberculosis  that  it  was  necessary  to  condemn  and  tank  their 
carcasses  under  the  Federal  meat-inspection  regulations.  This  evidence,  kindly 
presented  to  us  by  Doctor  Mohler,  raises  the  number  of  tuberculous  cattle  from 
which  blood  was  tested  to  50,  and  the  number  of  guinea  pigs  that  received 
injections  of  blood  from  tuberculous  cattle  without  contracting  tuberculosis 
to  135 

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